Obesity is a major disorder affecting as much as one third of the North American population. Several studies have shown that such individuals are at increased risk in developing cardiovascular disease (hypertension and hypercholesterolemia), diabetes and several types of cancer. The effective treatment of obesity, however, remains a largely unachieved goal. Existing pharmaco-therapeutic approaches to weight loss involve the use of amphetamine-based agents such as amphetamine, diethylpropion, mazindol and fenfluramine which act directly on the central nervous system (“CNS”) to lower food intake by modulating dopaminergic, adrenergic and/or serotonergic mechanisms. Although weight loss can be achieved with such agents, their use is restricted due to CNS side-effects, potential addiction liability and the production of tolerance to their actions, with chronic administration leading to potential depression, vestibular disturbances, hallucinations and addiction, as well as interference with the actions of other drugs, such as MAO inhibitors and antihypertensives. There is also a subpopulation of obese patients that is refractory to present anorectic drug treatments. The medical need is high for an effective anorectic agent that overcomes the above disadvantages of existing therapies. Of particular need are agents which act by alternative mechanisms to modulate food intake and/or metabolism.
Throughout this application, various publications are referenced. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the art to which this invention pertains.
Neuropeptide Y (“NPY”)
Neuropeptides are small peptides originating from large precursor proteins synthesized by peptidergic neurons and endocrine/paracrine cells. They hold promise for treatment of neurological, psychiatric, and endocrine disorders (De Wied, D. In: Neuropeptides: Basics and Perspectives (Elsevier, Amsterdam-New York-Oxford), 1990). Often the precursors contain multiple biologically active peptides. There is great diversity of neuropeptides in the brain caused by alternative splicing of primary gene transcripts and differential precursor processing. The neuropeptide receptors serve to discriminate between ligands and to activate the appropriate signals. Thus, it is expected that the receptors for neuropeptides consist of a large number of members.
Neuropeptide Y (NPY), a 36-amino acid peptide, is the most abundant neuropeptide to be identified in mammalian brain. Human NPY has the formula: H-Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu-Ile-Thr-Arg-Gln-Arg- Tyr-NH2 (SEQ. ID. NO. 1). Porcine and rat NPY have the same sequence except for Leu instead of Met in the 17-position.
NPY is an important regulator in both the central and peripheral nervous systems (Heilig, M. and E. Widerlov. Neuropeptide Y: an overview of central distribution, functional aspects, and possible involvement of neuropsychiatric illnesses. Acta Psychiatr. Scand. 82:95-114 (1990)) and influences a diverse range of physiological parameters, including effects on psychomotor activity, food intake, central endocrine secretion, and vasoactivity in the cardiovascular system. High concentrations of NPY are found in the sympathetic nerves supplying the coronary, cerebral, and renal vasculature and has contributed to vasoconstriction. NPY binding sites have been identified in a variety of tissues, including spleen (Lundberg, J. M., A. Hemsen, O. Larsson, A. Rudehill, A. Saria, and B. Fredholm. Neuropeptide Y receptor in pig spleen: binding characteristics, reduction of cyclic AMP formation and calcium antagonist inhibition of vasoconstriction. Eur. J. Pharmacol. 145:21-29 (1988)), intestinal membranes, brain (Hinson, J., C. Rauh, and J. Coupet. Neuropeptide Y stimulates inositol phospholipid hydrolysis in rat brain microprisms. Brain Response. 446:379-382 (1988)), aortic smooth muscle (Mihara, S., Y. Shigeri, and M. Fujimoto. Neuropeptide Y-induced intracellular Ca2+ increase in vascular smooth muscle cells. FEBS Lett. 259: 79-82 (1989)), kidney, testis, and placenta (Dumont, Y., J.C. Martel, A. Fournier, S. St.-Pierre, and R. Quiron. Neuropeptide Y and neuropeptide Y receptor subtypes in brain and peripheral tissues. Prog. Neurobiol. 38:125-167 (1992)). In addition, binding sites have been reported in a number of rat and human cell lines (e.g. Y1 in SK-N-MC, MC-IXC, CHP-212, and PC12 cells; Y2 in SK-N-Be(2), CHP-234, and SMS-MSN) (Grundemar, L., S. P. Sheikh, and C. Wahlestedt, In: The Biology of Neuropeptide Y and Related Peptides. (Humana Press, Inc., Totawa, N.J.), (1992)).
NPY forms a family (called the pancreatic polypeptide family) together with pancreatic polypeptide (PP) and peptide YY (PYY) which all consist of 36 amino acids and have a common tertiary structure, the so-called PP-fold (Glover, I. D., D. J. Barlow, J. E. Pitts, S. P. Wood, I. J. Tickle, T. L. Blundell, K. Tatemoto, J. R. Kimmel, A. Wollmer, W. Strassburger, and Y.-S. Zhang. Conformational studies of the pancreatic polypeptide hormone family. Eur. J. Biochem. 142:379-385 (1985)). Specific features of this family include a polyproline helix in residues 1 through 8, beta-turn in residues 9 through 14, an alpha-helix in residues 15 through 30, an outward-projecting C-terminus in residues 30 through 36, and a carboxy terminal amide (Schwartz, T. W., J. Fuhlendorff, L. L. Kjems, M. S. Kristensen, M. Vervelde, M. O'Hare, J. L. Krstenansky, and B. Bjornholm. Signal epitopes in the three-dimensional structure of neuropeptide Y. Ann. N.Y. Acad. Sci. 611:35-47 (1990)). The C-terminal amidated residue of these peptides appears to be essential for biological activity (Wahlestedt et al., 1986). Studies with peptide fragments of NPY have indicated that multiple NPY receptor subtypes exist (Wahlestedt, C., N. Yanaihara, and R. Hakanson. Evidence for different pre- and postjunctional receptors for neuropeptide Y and related peptides. Regul. Pept. 13:307-318 (1986)). Specifically, six receptor subtypes, denoted as Y1, Y2, Y3, Y4, Y5, and Y6, are understood to mediate the actions of NPY with each to-date, except for Y3, having been cloned.
The Y1, Y2, Y4, and Y5 receptors have been proposed to regulate feeding behavior, i.e. food intake, in subjects. A key pharmacological feature which distinguishes Y1 from Y2 is the fact that the Y1 receptor (and not the Y2 receptor) responds to an analog of NPY modified at residues 31 and 34 ([Leu31,Pro34]NPY), whereas the Y2 receptor (and not the Y1 receptor) has high affinity for the NPY peptide carboxyl-terminal fragment NPY-(13-36)(Fuhlendorff, J., U. Gether, L. Aakerlund, N. Langeland-Johansen, H. Thogersen, S. G. Melberg, U. B. Olsen, O. Thastrup, and T. W. Schwartz. [Leu31,Pro34]Neuropeptide Y: A specific Y1 receptor agonist. Proc. Natl. Acad. Sci. USA 87:182-186 (1990)). NPY analogs and N-terminally-shortened fragments, e.g. NPY(18-36), which contain one or more specific D-isomer substitutions for the naturally occurring residues (as well as pharmaceutically acceptable salts thereof), dispersed in a pharmaceutically acceptable liquid or solid carrier, can be administered to mammals, including humans, to substantially lower blood pressure over an extended period of time or to counteract hypertension.
Experimental and clinical observations also have supported the concept that neuropeptides play central roles in neurotransmission as well as the regulation of secretory functions of adenohypophysial, pancreatic, adrenalcortical and gut cells. Among the thirty or so neuropeptides that have been implicated in neuronal function in the mammalian central nervous system, several have also been suggested to function as neurotransmitters or neuromodulators primarily in afferent neurons.
An additional action of NPY is to decrease cardiac contractility (inotropy). This is an extremely important action of NPY, because it is known that, under many circumstances in which inotropy is decreased, diseases of life-threatening importance, e.g. congestive heart failure and cardiogenic shock, are associated with probable increased release of NPY into the blood. Prevention of NPY release, using a presynaptic NPY agonist, or NPY's action, using a postsynaptic NPY antagonist, may be beneficial in these disease states.
NPY has also been reported to produce coronary artery vasoconstriction and thereby may decrease myocardial blood flow resulting in myocardial ischemia. Such a circumstance can result in angina pectoris or, under more severe circumstances, may result in myocardial infarction and death. In recent years, several classes of drugs have proven effective in dilating coronary arteries to prevent such events.
Peptide YY (“PYY”)
Peptide YY (PYY) is a 36-residue peptide amide isolated originally from porcine intestine, and localized in the endocrine cells of the gastrointestinal tract and pancreas (Tatemoto et al. Proc. Natl. Acad. Sci. 79:2514, 1982). Peptide YY has N-terminal and C-terminal tyrosine amides; accordingly, these two tyrosines give PYY its name (Y represents the amino acid tyrosine in peptide nomenclature). In addition, PYY shares a number of central and peripheral regulatory roles with its homologous peptide Neuropeptide Y (NPY), which was originally isolated from porcine brain (Tatemoto, Proc. Natl. Acad. Sci. 79:5485, 1982). PYY is localized in intestinal cells; NPY, in contrast, is present in the submucous and myenteric neurons that innervate the mucosal and smooth muscle layers, respectively (Ekblad et al. Neuroscience 20:169, 1987). Both PYY and NPY are believed to inhibit gut motility and blood flow (Laburthe, Trends Endocrinol. Metab. 1:168, 1990), and they are also thought to attenuate basal (Cox et al. Br. J. Pharmacol. 101:247, 1990; Cox et al. J. Physiol. 398:65, 1988; Cox et al. Peptides 12:323, 1991; Friel et al. Br. J. Pharmacol. 88:425, 1986) and secretatogue-induced intestinal secretion in rats (Lundberg et al. Proc. Natl. Acad. Sci. USA 79:4471, 1982; Playford et al. Lancet 335: 1555, 1990) and humans (Playford et al., supra), as well as stimulate net absorption (MacFadyen et al. Neuropeptides 7:219, 1986). Elevated plasma PYY levels have been reported in individuals suffering from several conditions that cause diarrhea (Adrian et al. Gastroenterology 89:1070, 1985). Taken together, these observations suggest that PYY and NPY are released into the circulation after a meal (Adrian et al. Gastroenterology 89:1070, 1985: Balasubramaniam et al. Neuropeptides 14:209, 1989), and, thus may play a physiological role in regulating intestinal secretion and absorption, serving as natural inhibitors of diarrhea.
A high affinity PYY receptor system that exhibits a slightly higher affinity for PYY than NPY has been characterized in rat intestinal epithelia (Laburthe et al. Endocrinology 118:1910, 1986; Laburthe, Trends Endocrinol. Metabl. supra) and shown to be negatively coupled to adenylate cyclase (Servin et al. Endocrinology 124:692, 1989). Consistently, PYY exhibited greater antisecretory potency than NPY in voltage clamped preparations of rat small intestine (Cox et al. J. Physiol. supra), while C-terminal fragments of NPY were found to be less effective in their antisecretory potency than PYY (Cox et al. Br. J. Pharmacol, supra). Structure-activity studies using several partial sequences have led to the identification of PYY(22-36) as the active site for interacting with intestinal PYY receptors (Balasumbramaniam et al. Pept. Res. 1:32, 1988). This intestinal PYY-preferring receptor has now been cloned and shown to be identical to the Y2 receptors cloned from the brain (Goumain et al. Mol Pharmacol 60:124-134, 2001).
In addition, PYY has been implicated in a number of physiological activities including nutrient uptake (see, e.g., Bilcheik et al. Digestive Disease Week 506:623, 1993), cell proliferation (see, e.g., Laburthe, Trends Endocrinol. Metab. 1:168, 1990; Voisin et al. J. Bio. Chem., 1993), lipolysis (see, e.g., Valet et al. J. Clin. Invest. 85:291, 1990), and vasoconstriction (see, e.g., Lundberg et al., Proc. Natl. Acad. Sci, USA 79:4471, 1982).
The amino acid sequences of porcine and human PYY are as follows: porcine PYY: YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY (SEQ. ID. NO. 2), human PYY: YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY (SEQ. ID. NO. 3). The amino acid sequences for dog PYY and for rat PYY are the same as that of porcine PYY.
With respect to PYY, it has been reported previously that peripheral administration of PYY(3-36), a NPY Y2-preferring ligand, can on peripheral administration attenuate food intake in normal and fasted mice and rats as well as in normal and obese humans (Nature 418:650-654; 2002, N Engl J Med 349:941-948; 2003). The anorexigenic actions of PYY(3-36) are suggested to be mediated by arcuate nucleus Y2 receptors. One advantage of using Y2 selective ligands is that they can suppress the food intake on peripheral administration, whereas Y1 and Y5 selective ligands, as described above, have to penetrate the BBB to modulate food intake.
In addition to interacting with the Y2 receptor, PYY(3-36) can potently activate Y4 and Y5 receptors. Consequently, the inventor previously developed Y2 receptor selective agonists that are based on PYY(22-36) and PYY(25-36) (See U.S. Pat. Nos. 5,604,203, and 6,046,167 to Balasubramaniam) which are devoid of activities at the other NPY receptors including Y1, Y4, and Y5 at concentrations up to 20,000 nM. Preferred PYY(25-36) analogs include N-α-Ac-[Trp30]PYY(25-36)-NH2 (SEQ. ID. NO. 4), referred to as BWX-115, N-α-Ac-[Trp27, ψ35/36]PYY(25-36)-NH2 (SEQ. ID. NO. 5), referred to as BT-56, and N-α-Ac-[Trp30, ψ35/36]PYY(25-36)-NH2 (SEQ. ID. NO. 6), referred to as BT-123, and the PYY(22-36) analog N-α-Ac[Nle24,28, Trp30, Nva31, ψ35/36]PYY(22-36)-NH2 (SEQ. ID. NO. 7), referred to as B-48, [wherein ψ in the foregoing formulas is —CH2-NH—] which can be used to control food intake in animals and humans.
Pancreatic Peptide (“PP”)
Pancreatic peptide (PP) is a 36-amino-acid secretory peptide that is predominantly produced by the pancreas and released in response to nutrient stimuli.
The amino acid sequence of human PP is as follows:
(SEQ. ID. NO. 8)APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY.
PP, like PYY, shares a number of central and peripheral regulatory roles with its homologous peptide NPY. It has been reported recently that PP can attenuate body weight increase in mice through inhibiting food intake and increasing energy expenditures (Gastroenterology 124:1325-1336; 2003). These actions are mediated by altering the expression of orexigenic (NPY, orexin & ghrelin downregulated) and anorexigenic (urocortin upregulated) peptides, and decreasing gastric emptying and activity of the vagovagal or vago-sympathetic reflex arc. In addition, the anorectic effects of PP have been suggested to be mediated by the Y4 receptors in the area postrema (AP) because: a) autoradiography following peripheral administration of PP revealed high accumulation of PP in AP; b) expression of c-fos is seen in AP following iv administration of PP; and c) high density of Y4 receptors are present in AP (Brain Res 760:137-149; 1997).
One advantage of using Y4 selective ligands, like the Y2 selective ligand, is that they can suppress the food intake on peripheral administration, whereas Y1 and Y5 selective ligands, as described above, have to penetrate the BBB to modulate food intake. Native PP may not be an ideal candidate for suppressing food intake because of its short half-life of about six minutes and its interactions with Y5 receptors.
Accordingly, it would be desirable to develop NPY Y4 receptor agonists that can be used alone or mixed with NPY Y2 receptor agonists, as disclosed in U.S. Pat. Nos. 5,604,203, and 6,046,167 to Balasubramaniam, which are herein incorporated by reference, for controlling an NPY mediated physiological response in a subject, such as to control food intake in mammals, and that are expected to prove useful in the treatment of weight problems (e.g. obesity and diabetes), eating disorders, and such.